Highly unsaturated fatty acid or highly unsaturated fatty acid ethyl ester with reduced environmental pollutants, and method for producing same

ABSTRACT

A highly unsaturated fatty acid or a highly unsaturated fatty acid ethyl ester that has been produced using as a feedstock oil a fat or oil that contains highly unsaturated fatty acids as constituent fatty acids and which has been reduced in the contents of environmental pollutants, wherein among the dioxins contained, polychlorinated dibenzoparadioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are contained in amounts of less than 0.05 pg-TEQ/g and coplanar PCBs (Co-PCBs) in amounts of less than 0.03 pg-TEQ/g. Also disclosed is a method for producing the highly unsaturated fatty acid or highly unsaturated fatty acid ethyl ester by the steps of removing free fatty acids and environmental pollutants by thin-film distillation from a feedstock oil, ethyl esterifying the resulting fat or oil, and refining the same by rectification and column chromatography.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 17/317,179 filed on May 11, 2021, which is a continuation ofU.S. patent application Ser. No. 16/514,191 filed on Jul. 17, 2019 andissued as U.S. Pat. No. 11,034,643 on Jun. 15, 2021, which is acontinuation of U.S. patent application Ser. No. 15/366,522, filed onDec. 1, 2016 and issued as U.S. Pat. No. 10,399,922 on Sep. 3, 2019,which is a division of U.S. patent application Ser. No. 14/400,652,filed on Nov. 12, 2014 and issued as U.S. Pat. No. 9,540,306 on Jan. 10,2017, which is a U.S. national stage of application No.PCT/JP2013/063425, filed on May 14, 2013, which claims priority under 35U.S.C. §119(a) and 35 U.S.C. §365(b) to Japanese Application No.2012-110809, filed on May 14, 2012, the disclosures of each of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a method for reducing the quantities ofenvironmental pollutants, in particular, dioxins, brominated flameretardants, and other substances contained in fats or oils containingthe highly unsaturated fatty acids in the production of highlyunsaturated fatty acids or highly unsaturated fatty acid ethyl estersfrom the fats or oils. The present invention further relates to foods,supplements, medicines, cosmetics and feeds that are produced from thefats or oils prepared in accordance with such methods.

BACKGROUND ART

Environmental pollutants typified by dioxins are found almost everywhereon Earth today. They are also known to exert effects on fishes living inpolluted ocean areas. They are considered to have no direct effects onhealth if present in trace amounts but, nevertheless, the ingredients tobe ingested by humans as foods or feeds desirably contain the leastamounts of environmental pollutants.

Marine product oils, for example, fish oils contain highly unsaturatedfatty acids such as EPA (eicosapentaenoic acid, C20:5, n-3,all-cis-5,8,11,14,17-eicosdapentaenoic acid) and DHA (docosahexaenoicacid, C22:6, n-3, all-cis-4,7,10,13,16,19-docosahexaenoic acid). Beingknown to have various physiological functions, EPA and DHA are used asingredients of medicines, health foods, foods in general, feeds and thelike. Various purification steps are applied to make use of the EPAand/or DHA in marine product oils.

Non-Patent Document 1 discloses removing the insecticide DDT and itsmetabolites from fish oils by molecular distillation. Non-PatentDocument 2 discloses that chlorinated hydrocarbons and free fatty acidscan be removed from fats or oils using vacuum stripping or thin-filmdistillation. Non-Patent Document 3 discloses using physical refiningand molecular distillation in order to remove free fatty acids and otherundesired substances from oil compositions.

Patent Document 1 and Non-Patent Document 4 each disclose a method forreducing the quantities of environmental pollutants in a mixturecontaining fats or oils which comprises the step of adding a volatileworking fluid to the mixture and the step of subjecting the mixture toat least one stripping process together with the added volatile workingfluid.

Non-Patent Document 5 discloses that highly unsaturated fatty acids aredecomposed thermally when fish oils are deodorized at high temperatures.

Non-Patent Document 6 discloses removing dioxins, free fatty acids andcholesterols from fish oils by short path distillation so as to makethem suitable for use as feedstocks for ethyl esters.

CITATION LIST Patent Literature

Patent Document 1: Japanese Patent No. 3905538 (W02004/007654)

Non-Patent Literature

Non-Patent Document 1: K. Julshamn, L. Karlsen and O. R. Braekkan,Removal of DDT and its metabolites from fish oils by moleculardistillation, Fiskeridirektoratetsskrifter; Serie teknologiskeundersokelser, Vol. 5, No. 15 (1973)

Non-Patent Document 2: Anthony P. Bimbo: Guidelines for characterizationof food-grade fish oil. INFORM 9 (5), 473-483 (1998)

Non-Patent Document 3: Jiri Cmolik, Jan Pokorny: Physical refining ofedible oils, Eur. J. Lipid Sci. Technol. 102 (7), 472-486 (2000)

Non-Patent Document 4: Harald Breivik, Olav Thorstad: Removal of organicenvironmental pollutants from fish oil by short-path distillation, LipidTechnology, 17 (3), 55-58 (2005)

Non-Patent Document 5: Veronique Fournier et al.; Thermal degradation oflong-chain polyunsaturated fatty acids during deodorization of fishoil., Eur. J. Lipid Sci. Technol., 108, 33 -42 (2006)

Non-Patent Document 6: YUSHI, 62(11), 38-39, 2009

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a method for reducingthe quantities of environmental pollutants, in particular, dioxins andbrominated flame retardants, contained in fats or oils that contain thehighly unsaturated fatty acids as constituent fatty acids in theproduction of ethyl esters of highly unsaturated fatty acids from thefats or oils; another object of the present invention is to provideethyl esters having smaller contents of the dioxins and brominated flameretardants.

Solution To Problem

The present invention has been completed on the basis of the findingthat if fats or oils in which those highly unsaturated fatty acids thatare prone to deteriorate as through oxidation or isomerization arecontained as constituent fatty acids are refined by performing moleculardistillation or short path distillation under specified conditions, theconcentrations of environmental pollutants can be reduced to very lowlevels while suppressing the deterioration of the highly unsaturatedfatty acids. According to this method, the quantities of environmentalpollutants in the fats or oils can be reduced in such a way that thetotal content of dioxins is less than 0.2 pg-TEQ/g where TEQ is shortfor toxicity equivalency quantity. Further in addition, ethyl esters canbe obtained using the thus processed fats or oils as a feedstock. Bysubjecting the thus obtained ethyl esters to distillation and columnchromatographic treatment, the quantities of dioxins in the ethyl esterscan be further reduced.

In essence, the present invention relates to the highly unsaturatedfatty acids or highly unsaturated fatty acid ethyl esters describedbelow and the methods of producing the same, as well as feeds, foods,medicines, etc. that contain the same.

(1) A highly unsaturated fatty acid or a highly unsaturated fatty acidethyl ester which has been produced using as a feedstock oil a fat oroil that contains highly unsaturated fatty acids as constituent fattyacids and which has been reduced in the contents of environmentalpollutants, wherein among the dioxins contained, polychlorinateddibenzoparadioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) arecontained in amounts of less than 0.05 pg-TEQ/g and coplanar PCBs(Co-PCBs) in amounts of less than 0.03 pg-TEQ/g.

-   (2) The highly unsaturated fatty acid or highly unsaturated fatty    acid ethyl ester according to (1), wherein the content of a    brominated flame retardant contained has been reduced to such a    level that the amount of BDE-47 is less than 0.18 ng/g, the amount    of BDE-100 is less than 0.03 ng/g, the amount of BDE-49 is less than    0.05 ng/g, or the amount of BDE-99 is less than 0.05 ng/g.-   (3) The highly unsaturated fatty acid ethyl ester according to (1)    or (2), wherein the concentration as occupied by highly unsaturated    fatty acids in the sum of fatty acids is at least 80 area %, at    least 85 area %, at least 9 0area %, at least 95 area %, or at least    96 area %.-   (4) The highly unsaturated fatty acid or highly unsaturated fatty    acid ethyl ester according to any one of (1) to (3), wherein the fat    or oil containing highly unsaturated fatty acids as constituent    fatty acids is fish oil, krill oil, marine mammal oil or    microorganism oil.-   (5) The highly unsaturated fatty acid or highly unsaturated fatty    acid ethyl ester according to any one of (1) to (4), wherein the    highly unsaturated fatty acid is any one of eicosapentaenoic acid,    docosahexaenoic acid, docosapentaenoic acid, dihomo-□-linoleic acid    and arachidonic acid or a combination thereof-   (6) A medicine, supplement or food which comprises the highly    unsaturated fatty acid or highly unsaturated fatty acid ethyl ester    according to any one of (1) to (5) as an active ingredient.-   (7) A method for producing a highly unsaturated fatty acid or a    highly unsaturated fatty acid ethyl ester with reduced contents of    polychlorinated dibenzoparadioxins (PCDDs), polychlorinated    dibenzofurans (PCDFs) and coplanar PCB (Co-PCBs) by the steps of:-   a) removing free fatty acids and environmental pollutants by    thin-film distillation from a feedstock oil containing highly    unsaturated fatty acids as constituent fatty acids;-   b) hydrolyzing or ethyl esterifying the resulting highly unsaturated    fatty acid containing fat or oil; and-   c) refining the same by rectification and column chromatography.-   (8) The method according to (7), wherein the content of a brominated    flame retardant is also reduced.-   (9) The method according to (7) or (8), wherein the concentration as    occupied by highly unsaturated fatty acids in the sum of fatty acids    in the highly unsaturated fatty acid or highly unsaturated fatty    acid ethyl ester is at least 80 area %, at least 85 area %, at least    90 area %, at least 95 area %, or at least 96 area %.-   (10) The method according to any one of (7) to (9), wherein the    feedstock oil containing highly unsaturated fatty acids as    constituent fatty acids is fish oil, krill oil, marine mammal oil or    microorganism oil.-   (11) The method according to any one of (7) to (10), wherein the    thin-film distillation is carried out at a temperature of 200-270°    C., 220-260° C., or 220-250° C.-   (12) The method according to any one of (7) to (11), wherein the    thin-film distillation is carried out at a pressure of 5 Pa or    lower, 2 Pa or lower, or 1 Pa or lower.-   (13) The method according to any one of (7) to (12), wherein the    thin-film distillation is carried out at a flow rate of 20-200    (kg/h)/m2 or 25-120 (kg/h)/m2.-   (14) The method according to any one of (7) to (13), wherein the    thin-film distillation is molecular distillation or short path    distillation.-   (15) The method according to any one of (7) to (14), wherein    rectification is performed in three or more distillation columns.-   (16) The method according to any one of (7) to (15), wherein    refining by column chromatography uses column chromatography of    reverse-phase distribution type.-   (17) A highly unsaturated fatty acid or a highly unsaturated fatty    acid ethyl ester that have been produced by the method according to    any one of (7) to (16) and whose content of dioxins is less than    0.07 pg-TEQ/g or 0.05 pg-TEQ/g.-   (18) The highly unsaturated fatty acid or highly unsaturated fatty    acid ethyl ester according to-   (17), wherein the content of a brominated flame retardant contained    has been reduced to such a level that the amount of BDE-47 is less    than 0.18 ng/g, the amount of BDE-100 is less than 0.03 ng/g, the    amount of BDE-49 is less than 0.05 ng/g, or the amount of BDE-99 is    less than 0.05 ng/g.-   (19) The highly unsaturated fatty acid or highly unsaturated fatty    acid ethyl ester according to-   (17) or (18), wherein the concentration as occupied by highly    unsaturated fatty acids in the sum of fatty acids is at least 80    area %, at least 85 area %, at least 90 area %, at least 95 area %,    or at least 96 area %.-   (20) A medicine, supplement or food which contains the highly    unsaturated fatty acid or highly unsaturated fatty acid ethyl ester    according to any one of (17) to (19).

ADVANTAGEOUS EFFECTS OF INVENTION

The method of the present invention ensures that by virtue ofdistillation at high temperatures in high vacuum over a short period oftime, the quantities of environmental pollutants, and dioxins inparticular, that are contained in fish oils and the like can be reducedto very low levels without affecting the percentage of highlyunsaturated fatty acids in the sum of fatty acids and, hence, it ispossible to provide feeds, foods, supplements, medicines and variousother products as prepared from fish oils and other feedstocks withwhich there is no need to worry about contamination by dioxins.

DESCRIPTION OF EMBODIMENTS

On the following pages, the present invention will be described indetail.

As used herein, the term “highly unsaturated fatty acids” refers tofatty acids containing at least 18 carbon atoms and at least 3 doublebonds, more preferably fatty acids containing at least 20 carbon atomsand at least 3 or 4 double bonds, and most preferably fatty acidscontaining at least 20 carbon atoms and at least 5 double bonds.Specific examples include α-linoleic acid (18:3, n-3), y-linoleic acid(18:3, n-6), dihomo-y-linoleic acid (20:3, n-6), arachidonic acid (20:4,n-6), eicosapentaenoic acid (20:5, n-3), docosapentaenoic acid (22:5,n-6), docosahexaenoic acid (22:6, n-3), etc.

These are known to be abundant in certain kinds of microorganism oils,vegetable oils, and marine animal oils. Specific examples include: fishoils such as sardine oil, tuna oil, bonito oil, Men Hayden oil, codliver oil, herring oil, capelin oil, and salmon oil; marine animal oilsas from crustaceans such as krill; vegetable oils as from perilla, flax,soybean, and rapeseed; fats or oils produced by microorganisms belongingto the genus Mortierella, the genus Penicillium, the genus Aspergillus,the genus Rhodotorula, and the genus Fusarium.

The method of the present invention is suitable for application to fatsor oils derived from marine products with which there are particularconcerns about contamination by dioxins, as exemplified by fish oils,krill oil, or marine mammal oils.

As used herein, the term “fats or oils containing highly unsaturatedfatty acids as constituent fatty acids” means triglycerides orphospholipids.

If these fats or oils are to be used as feedstock oils in the presentinvention, preliminary treatments may be performed before they aresubjected to molecular distillation or short path distillation. Thepreliminary treatments may be exemplified by a degumming step, adecoloring step using activated clay or activated charcoal, and awashing step with water.

As used herein, the term “environmental pollutants” embraces:polychlorinated biphenyls (PCBs), DDTs, polychlorinated triphenyls(PCTs), dibenzo-dioxins (PCDDs), and dibenzo-furans (PCDFs);chlorophenols and hexachlorocyclohexanes (HCHs), toxaphenes, dioxins,brominated flame retardants, polyaromatic hydrocarbons (PAHs), organotincompounds (e.g. tributyltin and triphenyltin), organomercury compounds(e.g. methylmercury), etc. To give a guide figure for the degree bywhich these environmental pollutants have been removed, the sum ofdioxins which are representative of the ubiquitous anddifficult-to-remove substances is designated in terms of toxicequivalency quantity (pg-TEQ/g).

As used herein, the term “dioxins” refers to the sum of thepolychlorinated dibenzoparadioxins (PCDDs), polychlorinateddibenzofurans (PCDFs), and coplanar PCBs (Co-PCBs) that are listed inTable 1 below; the contents of the respective members were measured,each measured value was multiplied by the corresponding toxicityequivalency factor and summed up to calculate the toxicity equivalencyquantity (pg-TEQ/g).

Measurements were also made of brominated flame retardants. The term“brominated flame retardants” collectively refers to the compoundslisted in Table 9 below. Although contamination by dioxins in theenvironment has recently begun to decrease, contamination by brominatedflame retardants still tends to increase and hence deserves specialnote. Applicable indicators are BDE-100, BDE-49, BDE-99, and BDE-47which are relatively high amount in fish oils.

TABLE 1 Items for Analysis No. of Cl atoms Items for AnalysisAbbreviation PCDD 4 2,3,7,8-tetrachlorodibenzo-p-dioxin 2,3,7,8-TeCDDTetrachlorodibenzo-p-dioxins in total TeCDDs 51,2,3,7,8-pentachlorodibenzo-p-dioxin 1,2,3,7,8-PeCDDPentachlorodibenzo-p-dioxins in total PeCDDs 61,2,3,4,7,8-hexachlorodibenzo-p-dioxin 1,2,3,4,7,8-HxCDD1,2,3,6,7,8-hexachlorodibenzo-p-dioxin 1,2,3,6,7,8-HxCDD1,2,3,7,8,9-hexachlorodibenzo-p-dioxin 1,2,3,7,8,9-HxCDDHexachlorodibenzo-p-dioxins in total HxCDDs 71,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin 1,2,3,4,6,7,8-HpCDDHeptachlorodibenzo-p-dioxins in total HpCDDs 8Octachlorodibenzo-p-dioxin OCDD PCDF 4 2,3,7,8-tetrachlorodibenzofuran2,3,7,8-TeCDF Tetrachlorodibenzofurans in total TeCDFs 51,2,3,7,8-pentachlorodibenzofuran 1,2,3,7,8-PeCDF2,3,4,7,8-pentachlorodibenzofuran 2,3,4,7,8-PeCDFPentachlorodibenzofurans in total PeCDFs 61,2,3,4,7,8-hexachlorodibenzofuran 1,2,3,4,7,8-HxCDF1,2,3,6,7,8-hexachlorodibenzofuran 1,2,3,6,7,8-HxCDF1,2,3,7,8,9-hexachlorodibenzofuran 1,2,3,7,8,9-HxCDF2,3,4,6,7,8-hexachlorodibenzofuran 2,3,4,6,7,8-HxCDFHexachlorodibenzofurans in total HxCDFs 71,2,3,4,6,7,8-heptachlorodibenzofuran 1,2,3,4,6,7,8-HpCDF1,2,3,4,7,8,9-heptachlorodibenzofuran 1,2,3,4,7,8,9-HpCDFHeptachlorodibenzofurans in total HpCDFs 8 Octachlorodibenzofuran OCDFCo-PCB Non-ortho form 4 3,4,4′,5-tetrachlorobiphenyl 3,4,4′,5-TeCB (#81)3,3′,4,4′-tetrachlorobiphenyl 3,3′,4,4′-TeCB (#77) 53,3′,4,4′,5-pentachlorobiphenyl 3,3′,4,4′,5-PeCB (#126) 63,3′,4,4′,5,5′-hexachlorobiphenyl 3,3′,4,4′,5,5′-HxCB (#169) Mono-orthoform 5 2′,3,4,4′,5-pentachlorobiphenyl 2′,3,4,4′,5-PeCB (#123)2,3′,4,4′,5-pentachlorobiphenyl 2,3′,4,4′,5-PeCB (#118)2,3,3′,4,4′-pentachlorobiphenyl 2,3,3′,4,4′-PeCB (#105)2,3,4,4′,5-pentachlorobiphenyl 2,3,4,4′,5-PeCB (#114) 62,3′,4,4′,5,5′-hexachlorobiphenyl 2,3′,4,4′,5,5′-HxCB (#167)2,3,3′,4,4′,5-hexachlorobiphenyl 2,3,3′,4,4′,5-HxCB (#156)2,3,3′,4,4′,5′-hexachlorobiphenyl 2,3,3′,4,4′,5′-HxCB (#157) 72,3,3′,4,4′,5,5′-heptachlorobiphenyl 2,3,3′,4,4′,5,5′-HpCB (#189)

In the present invention, dioxins are removed by thin-film distillation,preferably by molecular distillation or short path distillation (SPD).Among applicable thin-film distillation methods, one that is performedwith the condenser placed within a shorter distance than the mean freepath of the vapor molecules evaporating from the heating surface at aspecified pressure under a high vacuum (<0.1 Pa) is called moleculardistillation. Short path distillation has been developed with a view toenhancing the distilling performance of molecular distillation. Shortpath distillation is performed at pressures in a medium vacuum regionhigher than 0.1 Pa, with the condenser placed at distances approximatelyequal to the mean free path of the evaporating molecules, so it is apractical method that is by far improved in distilling performance thanmolecular distillation.

In molecular distillation or short path distillation, the feed to bedistilled is exposed to high temperatures for such a short period oftime that both methods are adapted to removal of unwanted componentsfrom triglycerides containing EPA, DHA and other heat-sensitivesubstances.

In the present invention, molecular distillation or short pathdistillation is carried out at a temperature of 200-270° C., preferably220-260° C., and more preferably 220-250° C. Both types of distillationare carried out at a pressure lower than 5 Pa, preferably lower than 2Pa, and more preferably lower than 1 Pa. They are performed by thethin-film method that is carried out at a flow rate of 20-200 (kg/h)/m2,preferably 25-120 (kg/h)/m2. If the flow volume is unduly small, theproductivity will drop, so it is preferred to flow the feedstock oil ina maximum removable volume while ensuring that dioxins are beingremoved. If distillation is performed under these conditions, highlyunsaturated fatty acids, although being heat-sensitive, will sufferlittle deterioration in quality.

By effecting distillation under these conditions, the quantities ofdioxins PCDDs and PCDFs could be reduced to below the limit ofmeasurement, or substantially zero. The value zero typically means lessthan 0.043 pg-TEQ/g as calculated from the numerical values of detectionlimit listed in Table 6; in other words, the quantities of dioxins PCDDsand PCDFs can be reduced to less than 0.05 pg-TEQ/g. The quantities ofcoplanar PCBs can also be reduced to less than 0.2 pg-TEQ/g, even toless than 0.1 pg-TEQ/g, or to less than 0.05 pg-TEQ/g, or to less than0.02 pg-TEQ/g, and even to less than 0.01 pg-TEQ/g.

As for the brominated flame retardants, if BDE-100, BDE-49, BDE-99 andBDE-47 are chosen as indicators, their quantities can be reduced to lessthan 0.05 μg/g, preferably less than 0.03 μg/g, and more preferably lessthan 0.02 μg/g.

Embodiments of the invention method are described below morespecifically.

The feedstock oil is preferably one that has been subjected to adegumming process as by washing with water. The washed feedstock oil isimmediately subjected to molecular distillation or short pathdistillation under the conditions described above, so that cholesterols,free fatty acids, environmental pollutants and the like are removed asdistillate fractions, yielding the residue containing triglycerides. Theresidue may be used either immediately or after being subjected to adecoloring process as with activated charcoal or activated clay or to adeodorizing process as by steam distillation. The refined fat or oilthus produced can be used as an ingredient in feeds, foods, orsupplements.

The above-mentioned residue may be used as a feedstock for producingethyl esters containing dioxins in reduced amounts.

To this end, ethyl alcohol and a catalyst or an enzyme are added to theresidue and reaction is performed to generate esters of the constituentfatty acids of the triglyceride and the ethyl alcohol. This step ofethyl esterification may be performed by any known method.

If necessary, the formed ethyl ester may be further refined. In order toincrease the purity of EPA ethyl ester or DHA ethyl ester, an additionalmethod such as molecular distillation, rectification or columnchromatography may be applied. Specifically, refining can be achieved bysuch methods as disclosed in JP H5-222392 (Family Patent EP0610506), JPH4-41457 (Family Patent EP0460917), JP H6-33088, etc.

Rectification is performed under a high vacuum with three or moredistillation columns and the main distillate consisting of the EPA ethylester and/or DHA ethyl ester is separated from an initial fraction ofthe higher volatility and a bottoms fraction of the lower volatility.The conditions of rectification are such that the temperature is in therange of 150-200° C., preferably 160-190° C., more preferably 170-190°C. and the pressure is in the range of 1-300 Pa, preferably 1-200 Pa,more preferably 1-133 Pa. It is preferred that the main distillate of160-190° C., preferably 170-190° C. is obtained with the degree ofvacuum being chosen at 1-133 Pa.

A particularly preferred method is by performing column chromatographyafter rectification. The present inventors found that as the result ofconcentrating EPA and DHA by rectification, the relative concentrationsof dioxins rose but that by means of subsequent column chromatography,they could be rendered lower than the levels prior to the rectification.While silica gel, ion-exchange resin, activated clay, silver nitrate,etc. may be used in column chromatography, it is particularly preferredto perform column chromatography of reverse-phase distribution type. Areverse-phase distribution system may be created by using, for example,an alkyl group bound silica packing material (e.g. ODS column), withwater, alcohols or ketones being used as solvent. Methanol is preferred.These solvents may be used either independently or in admixture.

By combining the above-described techniques of rectification and columnchromatography, highly unsaturated fatty acids can be concentrated whilereducing the concentrations of environmental pollutants. Theconcentrations of highly unsaturated fatty acids, say, EPA ethyl esterand/or DHA ethyl ester, can be increased to a purity of at least 80area%, or at least 85area %, or at least 90area %, or 95area %, or even atleast 96area % while, at the same time, among the dioxins, PCDDs andPCDFs can be reduced in content below their limits of measurement,namely, substantially zero (less than 0.043 pg-TEQ/g as calculated fromthe detection limits) and the contents of coplanar PCBs can also belowered to less than 0.1 pg-TEQ/g, or less than 0.03 pg-TEQ/g, or evenless than 0.01 pg-TEQ/g. As for the brominated flame retardants, theirquantities can be lowered to less than 0.18 ng/g in terms of BDE-47, toless than 0.03 ng/g in terms of BDE-100, to less than 0.05 ng/g in termsof BDE-49, and to less than 0.05 ng/g in terms of BDE-99. The quantitiesof BDE-100, BDE-49, BDE-99 and BDE-47 can be lowered to less than 0.05ng/g, preferably less than 0.03 ng/g, more preferably less than 0.02μg/g. For applications as medicines, the concentration of EPA ethylester and/or DHA ethyl ester is preferably equivalent to a purity of atleast 96area %.

The above-described free fatty acids and fatty acid esters that havehigh concentrations of highly unsaturated fatty acids, but lowconcentrations of environmental pollutants are suitable for use asmaterials to prepare medicines and supplements that contain the highlyunsaturated fatty acids as an active ingredient.

In the case of use for the preparation of medicines and dietarysupplements, the EPA and/or DHA in fatty acids need be concentrated to ahigher content depending on the need. In that case, the highlyunsaturated fatty acids in the glyceride may be concentrated by a methodfor selective concentration of highly unsaturated fatty acids throughlipase reaction (as disclosed in WO2009/17102). Even the thus processedglyceride may be treated by the method of the present invention to havehigher concentrations of highly unsaturated fatty acids but lowerquantities of environmental pollutants.

The highly unsaturated fatty acid esters produced by the methoddescribed above may be hydrolyzed to give the highly unsaturated fattyacids.

On the following pages, Examples of the present invention are describedbut they are by no means intended to limit the scope of the presentinvention.

Measurements of Dioxins and Brominated Flame Retardants

In the following Examples of the present invention, a measurement ofdioxins was commissioned to JAPAN FOOD RESEARCH LABORATORIES. The methodof the measurement was in accordance with the “Provisional Guidelines onthe Methods of Measuring Dioxins in Foods (February 2008)” (EISHOKU No.138 and EINYU No. 200 in 1999).

A measurement of brominated flame retardants was commissioned to“eurofins”, a bio-analytical testing company. The method of themeasurement was in accordance with high-resolution mass spectrometry(HRGC/HRMS).

Measurement of Acid Value (AV)

In the Examples of the present invention, a measurement of acid value(AV) was conducted in accordance with the JOCS Standard Methods for theAnalysis of Fats, Oils and Related Materials (2003 Edition) (compiled byJapan Oil Chemists' Society).

Determining the Composition of Fatty Acids

The composition of fatty acids in a feedstock fish oil and that of fattyacids in the oil resulting from subsequent short path distillation weredetermined by gas chromatography after ethyl esterification of the fishoil. To be specific, 1 mL of 1 N sodium ethylate/ethanol solution wasadded to 40 μL of fish oil and the resulting mixture was stirred forabout 30 seconds. Subsequently, 1 mL of 1 N hydrochloric acid was addedto neutralize the mixture, to which 2 mL of hexane and 3 mL of asaturated aqueous solution of ammonia sulfate were added; after allowingthe stirred mixture to settle undisturbed, the upper layer was subjectedto measurement by gas chromatography.

Conditions for Gas Chromatographic Analysis

-   Device type: Agilent 6850 GC system (Agilent Technologies)-   Column: DB-WAX J&W 123-7032E-   Column temperature: 200° C.-   Injection temperature: 300° C.-   Injection method: Splitting-   Splitting ratio: 100:1-   Detector temperature: 300° C.-   Detector: FID-   Carrier gas: Helium

EXAMPLE 1

Two kinds of crude sardine oil different in acid value and dioxinscontent were distilled under various conditions to test for the removalof dioxins. The distillation apparatus used was a short pathdistillation (SPD) device KD 10 (product of UIC GmbH with a distillationsurface area of 0.1 m2). The distillation conditions (temperature,pressure, and flow rate) were as shown in Table 2. With the distillationtemperature fixed at 250° C., the pressure and flow rate were variedwithin the ranges of 0.4-3.0 Pa and 25-121 (kg/hr)/ m², respectively.

The dioxins concentrations and acid values of the crude oils and theproducts of distillation are shown in Table 2.

Irrespective of whether the crude oils had high or low acid values andalso irrespective of whether they contained dioxins in large or smallquantities, the contents of dioxins could be reduced to less than 0.1pg-TEQ/g by distillation under all conditions employed.

TABLE 2 Feedstock Crude sardine oil 1 Crude sardine oil 2 FeedstockCondition Feedstock Condition oil 1 1 2 3 oil 2 4 5 6 PCDD + PCDF(pg-TEQ/g) 0.20 0.00 0.00 0.00 3.48 0.00 0.00 0.00 Coplaner PCBs(pg-TEQ/g) 3.20 0.00 0.01 0.03 11.12 0.04 0.01 0.07 Total (of dioxins)(pg-TEQ/g) 3.40 0.00 0.01 0.03 14.60 0.04 0.01 0.08 Temperature ° C. 250249 249 249 249 249 Pressure Pa 0.7 1.3 1.5 0.4 0.8 3.0 Flow rate(kg/h)/m² 25 78 112 30 72 121 Acid value 10.51 0.29 0.38 0.35 4.86 0.120.26 0.21

EXAMPLE 2

Using the same apparatus as in Example 1, dioxins were removed from semirefined tuna oil (refined by degumming and deacidification) bydistillation under the conditions listed in

Table 3. As Table 3 shows, the contents of dioxins in the tuna oil couldalso be reduced to less than 0.1 pg-TEQ/g by the method of the presentinvention (distilled at a temperature of 250° C., at a pressure of 0.1Pa, and at a flow rate of 48 (kg/hr)/ m²).

Using a different short path distillation (SPD) device KD 6 (product ofUIC GmbH with a distillation surface area of 0.06 m²), dioxins wereremoved from crude sardine oil. As Table 3 shows, the contents ofdioxins could also be reduced to less than 0.1 pg-TEQ/g by the method ofthe present invention (distilled at a temperature of 270° C., at apressure of 0.6 Pa, and at a flow rate of 20 (kg/hr)/ m²).

TABLE 3 Feedstock Lightly refined tuna oil Crude sardine oil 3 Deviceused KD-10 KD-60 Feedstock oil 3 Condition 7 Feedstock oil 4 Condition 8PCDD + PCDF (pg-TEQ/g) 1.72 0.00 0.11 0.00 Coplaner PCBs (pg-TEQ/g) 7.860.04 7.87 0.01 Total (of dioxins) (pg-TEQ/g) 9.58 0.04 7.98 0.01Temperature ° C. 250 270 Pressure Pa 0.1 0.6 Flow rate (kg/h)/m² 48 20Residue wt % 99.2 92.1 Distilled-off content wt % 0.8 7.9 Acid value — —5.79 0.14

EXAMPLE 3

Using a centrifugal, molecular distillation apparatus MS380 (product ofNIPPON SHARYO, with a distillation surface area of 0.11 m²), dioxinswere removed from crude sardine oils by distillation under theconditions listed in Table 4. As Table 4 shows, even the moleculardistillation device was capable of reducing the contents of dioxins asin Examples 1 and 2, more specifically to less than 0.2 pg-TEQ/g.

TABLE 4 Feedstock Crude sardine oil 4 Crude sardine oil 5 FeedstockCondition Feedstock Condition oil 5 9 10 oil 6 11 12 PCDD + PCDF(pg-TEQ/g) 0.17 0.10 0.03 0.09 0.05 0.00 Coplaner PCBs (pg-TEQ/g) 1.120.05 0.00 0.52 0.01 0.00 Total (of dioxins) (pg-TEQ/g) 1.29 0.15 0.030.61 0.07 0.00 Temperature ° C. 220 240 240 260 Pressure Pa 0.67 0.670.67 0.67 Flow rate (kg/h)/m² 182 182 182 182 Acid value 5.61 0.31 0.122.85 0.17 0.11

EXAMPLE 4

A crude sardine oil (with an acid value of 6 and containing 19% EPA and8% DHA) was washed with warm (85° C.) water (5% relative to the crudeoil) and subjected to short path distillation using a short pathdistillation (SPD) device KD1800 (product of UIC GmbH, with adistillation surface area of 18 m²). The distillation conditions were asfollows: degree of vacuum, 0.7-1 Pa; device temperature, 250° C.; feedvolume, ca. 2000 kg/H (flow rate: 110 (kg/h)/ m²).

The results of measurement of dioxins in oil are shown in Table 5 (forthe feedstock oil) and Table 6 (for the SPD oil). The total quantity ofdioxins could be reduced from 3.0 pg-TEQ/g to 0.014 pg-TEQ/g in terms oftoxicity equivalency quantity. The acid value of the SPD oil was lessthan 0.2 and there was no change in the composition of fatty acids fromthe crude oil.

TABLE 5 Feedstock sardine oil Sample lot: 10.09 g Lower limit ofToxicity Toxicity Found detection equivalency equivalency concentrationin sample factor quantity (TEQ) Items for analysis (pg/g) (pg/g) (TEF)(pg-TEQ/g) PCDD 2,3,7,8-TeCDD N.D. 0.01 1 0 1,2,3,7,8-PeCDD 0.03 0.01 10.03 1,2,3,4,7,8-HxCDD N.D. 0.03 0.1 0 1,2,3,6,7,8-HxCDD N.D. 0.02 0.1 01,2,3,7,8,9-HxCDD N.D. 0.03 0.1 0 1,2,3,4,6,7,8-HpCDD N.D. 0.03 0.01 0OCDD 0.08 0.05 0.0003 0.000024 Total PCDDs — — — 0.030024 PCDP2,3,7,8-TeCDF 0.32 0.01 0.1 0.032 1,2,3,7,8-PeCDF 0.05 0.01 0.03 0.0015(+1,2,3,4,8-PeCDF) 2,3,4,7,8-PeCDF 0.09 0.01 0.3 0.027 1,2,3,4,7,8-HxCDF0.02 0.02 0.1 0.002 (+1,2,3,4,7,9-HxCDF) 1,2,3,6,7,8-HxCDF N.D. 0.02 0.10 1,2,3,7,8,9-HxCDF N.D. 0.03 0.1 0 2,3,4,6,7,8-HxCDF N.D. 0.03 0.1 01,2,3,4,6,7,8-HpCDF N.D. 0.02 0.01 0 1,2,3,4,7,8,9-HpCDF N.D. 0.02 0.010 OCDF N.D. 0.05 0.0003 0 Total PCDFs — — — 0.0625 Total (PCDDs + PCDFs)— — — 0.092524 Co-PCB Non-ortho forms 3,4,4′,5-TeCB (#81) 0.75 0.020.0003 0.000225 3,3′,4,4′-TeCB (#77) 82 0.05 0.0001 0.00823,3′,4,4′,5-PeCB (#126) 28 0.04 0.1 2.8 3,3′,4,4′,5,5′-HxCB (#169) 1.90.02 0.03 0.057 Total Non-ortho forms 112.65 — — 2.865425 Mono-orthoforms 2′,3,4,4′,5-PeCB (#123) 19 0.4 0.00003 0.00057 2,3′,4,4′,5-PeCB(#118) 1400 2 0.00003 0.042 2,3,3′,4,4′-PeCB (#105) 510 0.8 0.000030.0153 2,3,4,4′,5-PeCB (#114) 17 0.6 0.00003 0.00051 2,3′,4,4′,5,5′-HxCB(#167) 130 0.8 0.00003 0.0039 2,3,3′,4,4′,5-HxCB (#156) 210 0.5 0.000030.0063 2,3,3′,4,4′,5′-HxCB (#157) 44 0.4 0.00003 0.001322,3,3′,4,4′,5,5′-HpCB (#189) 24 0.4 0.00003 0.00072 Total Mono-orthoforms 2354 — — 0.07062 Total Co-PCBs 2466.65 — — 2.936045 Total dioxins— — — 3.0 Congeners PCDD TeCDDs N.D. 0.01 — — PeCDDs 0.03 0.01 — —HxCDDs N.D. 0.03 — — HpCDDs 0.04 0.03 — — OCDD 0.08 0.05 — — Total PCDDs0.15 — — — PCDF TeCDFs 0.43 0.01 — — PeCDFs 0.14 0.01 — — HxCDFs 0.020.03 — — HpCDFs N.D. 0.02 — — OCDF N.D. 0.05 — — Total PCDFs 0.59 — — —Total (PCDDs + PCDFs) 0.74 — — — Notes: 1. “N.D.” refers to the casewhere the found concentration was less than the lower detection limit.2. Toxicity equivalency quantity (TEQ) was calculated as if a foundconcentration less than the lower detection limit was zero. 3. Toxicityequivalency factor (TEF) was in accordance with WHO (2006). 4.1,2,3,7,8-PeCDF and 1,2,3,4,7,8-HxCDF are cited in total values sincethey could not be isolated by SP-2331 column.

TABLE 6 Sardine SPD oil Sample lot: 10.04 g Lower limit of ToxicityToxicity Found detection Equivalency equivalency concentration in samplefactor quantity (TEQ) Items for analysis (pg/g) (pg/g) (TEF) (pg-TEQ/g)PCDD 2,3,7,8-TeCDD N.D. 0.01 1 0 1,2,3,7,8-PeCDD N.D. 0.01 1 01,2,3,4,7,8-HxCDD N.D. 0.03 0.1 0 1,2,3,6,7,8-HxCDD N.D. 0.02 0.1 01,2,3,7,8,9-HxCDD N.D. 0.03 0.1 0 1,2,3,4,6,7,8-HpCDD N.D. 0.03 0.01 0OCDD N.D. 0.05 0.0003 0 Total PCDDs — — — 0 PCDF 2,3,7,8-TeCDF N.D. 0.010.1 0 1,2,3,7,8-PeCDF N.D. 0.01 0.03 0 (+1,2,3,4,8-PeCDF)2,3,4,7,8-PeCDF N.D. 0.01 0.3 0 1,2,3,4,7,8-HxCDF N.D. 0.02 0.1 0(+1,2,3,4,7,9-HxCDF) 1,2,3,6,7,8-HxCDF N.D. 0.02 0.1 0 1,2,3,7,8,9-HxCDFN.D. 0.03 0.1 0 2,3,4,6,7,8-HxCDF N.D. 0.03 0.1 0 1,2,3,4,6,7,8-HpCDFN.D. 0.02 0.01 0 1,2,3,4,7,8,9-HpCDF N.D. 0.02 0.01 0 OCDF N.D. 0.050.0003 0 Total PCDFs — — — 0 Total (PCDDs + PCDFs) — — — 0 Co-PCBNon-ortho forms 3,4,4′,5-TeCB (#81) N.D. 0.02 0.0003 0 3,3′,4,4′-TeCB(#77) 0.23 0.05 0.0001 0.000023 3,3′,4,4′,5-PeCB (#126) 0.10 0.04 0.10.010 3,3′,4,4′,5,5′-HxCB (#169) 0.02 0.02 0.03 0.0006 Total Non-orthoforms 0.35 — — 0.010623 Mono-ortho forms 2′,3,4,4′,5-PeCB (#123) 1.60.4  0.00003 0.000048 2,3′,4,4′,5-PeCB (#118) 75 2   0.00003 0.002252,3,3′,4,4′-PeCB (#105) 18 0.8  0.00003 0.00054 2,3,4,4′,5-PeCB (#114)1.5 0.6  0.00003 0.000045 2,3′,4,4′,5,5′-HxCB (#167) 10 0.8  0.000030.00030 2,3,3′,4,4′,5-HxCB (#156) 13 0.5  0.00003 0.000392,3,3′,4,4′,5′-HxCB (#157) 2.8 0.4  0.00003 0.0000842,3,3′,4,4′,5,5′-HpCB (#189) 2.2 0.4  0.00003 0.000066 Total Mono-orthoforms 124.1 — — 0.003723 Total Co-PCBs 124.45 — — 0.014346 Total dioxins— — — 0.014 Congeners PCDD TeCDDs N.D. 0.01 — — PeCDDs N.D. 0.01 — —HxCDDs N.D. 0.03 — — HpCDDs N.D. 0.03 — — OCDD N.D. 0.05 — — Total PCDDs0 — — — PCDF TeCDFs N.D. 0.01 — — PeCDFs N.D. 0.01 — — HxCDFs N.D. 0.03— — HpCDFs N.D. 0.02 — — OCDF N.D. 0.05 — — Total PCDFs 0 — — — Total(PCDDs + PCDFs) 0 — — — Notes: 1. “N.D.” refers to the case where thefound concentration was less than the lower detection limit. 2. Toxicityequivalency quantity (TEQ) was calculated as if a found concentrationless than the lower detection limit was zero. 3. Toxicity equivalencyfactor (TEF) was in accordance with WHO (2006). 4. 1,2,3,7,8-PeCDF and1,2,3,4,7,8-HxCDF are cited in total values since they could not beisolated by SP-2331 column.

EXAMPLE 5

Crude sardine oil was subjected to short path distillation under thesame conditions as in Example 4 and the composition of fatty acids wasdetermined by the previously described method both before and after thedistillation. The results are shown in Table 7. For fatty acids that had18 or more carbon atoms with 3 or more double bonds, and which wereprone to oxidize, isomerize or otherwise deteriorate upon heating, theproportions of such fatty acids in the total fatty acids in thefeedstock oil and the SPD oil, as well as the changes in suchproportions are shown in the table. Each of the fatty acids underexamination suffered only a little change in content and even thosefatty acids with the greater number of double bonds did not experienceany particularly great variations. From these results, it was verifiedthat the method of the present invention is a good way to remove dioxinswithout causing deterioration of highly unsaturated fatty acids. InTable 7, ARA stands for arachidonic acid and DPA docosapentaenoic acid.

TABLE 7 C18:3 C18:3 C18:4 C20:4 n-6 n- 3 n-3 ARA n-3 EPA DPA DHAFeedstock oil (area %) 0.65 0.62 2.55 1.25 0.85 21.89 2.60 7.67 Afterdistillation (area %) 0.63 0.57 2.53 1.26 0.86 21.86 2.62 7.65 Relativechange (After −0.02 −0.05 −0.03 0.01 0.01 −0.03 0.02 −0.01 distillation− Feedstock oil)

EXAMPLE 6

An ethyl ester of EPA was produced from the distilled oil as prepared inExample 4.

The production procedure was as follows: the SPD oil was subjected toethanolysis reaction with ethyl alcohol in the presence of an alkalicatalyst to form an ethyl ester; after washing with warm water, theethyl ester was dried and rectified with a degree of vacuum of 13 Pa togive the main distillate (with a temperature of ca. 176° C.), which wastreated by HPLC using column of reverse-phase distribution type (ODS);the solvent was subsequently distilled off to yield an ethyl ester of97% pure EPA.

The contents of dioxins in this ethyl ester were measured by the samemethod as in Example 4 and the results are shown in Table 8. No dioxinswould be concentrated during the ethyl esterification and the subsequentrefining step; use of the feedstock treated as in Example 4 enabled theproduction of EPA's ethyl ester samples in which the total quantity ofdioxins was less than 0.07 pg-TEQ/g in terms of toxicity equivalencyquantity. The data in Table 8 are for the production from threedifferent lots of feedstock. It was verified that the method of thepresent invention enabled consistent production of EPA's ethyl estersamples in which the total quantity of dioxins was between 0.006 and0.021 pg-TEQ/g in terms of toxicity equivalency quantity. As for NDsbelow the detection limit, calculation was made by inserting numericvalues of detection limit and yet the increase was only 0.005 innumerical value, meaning that it is possible to produce EPA's ethylester samples with 0.011-0.026 pg-TEQ/g.

TABLE 8 Lot. 1 Lot. 2 Lot. 3 Found Toxicity Found Toxicity FoundToxicity Toxicity concen- equivalency concen- equivalency concen-equivalency equivalency tration quantity tration quantity trationquantity factor(TEF) (pg/g) (pg-TEQ/g) (pg/g) (pg-TEQ/g) (pg/g)(pg-TEQ/g) PCDD 2,3,7,8-TCDD 1 N.D. 0 N.D. 0 N.D. 0 1,2,3,7,8-PeCDD 1N.D. 0 N.D. 0 N.D. 0 1,2,3,4,7,8-HxCDD 0.1 N.D. 0 N.D. 0 N.D. 01,2,3,6,7,8-HxCDD 0.1 N.D. 0 N.D. 0 N.D. 0 1,2,3,7,8,9-HxCDD 0.1 N.D. 0N.D. 0 N.D. 0 1,2,3,4,6,7,8-HpCDD 0.01 N.D. 0 N.D. 0 N.D. 0 OCDD 0.0003N.D. 0 N.D. 0 N.D. 0 PCDF 2,3,7,8-TCDF 0.1 N.D. 0 N.D. 0 N.D. 01,2,3,7,8-PeCDF 0.03 N.D. 0 N.D. 0 N.D. 0 2,3,4,7,8-PeCDF 0.3 N.D. 0N.D. 0 N.D. 0 1,2,3,4,7,8-HxCDF 0.1 N.D. 0 N.D. 0 N.D. 01,2,3,6,7,8-HxCDF 0.1 N.D. 0 N.D. 0 N.D. 0 1,2,3,7,8,9-HxCDF 0.1 N.D. 0N.D. 0 N.D. 0 2,3,4,6,7,8-HxCDF 0.1 N.D. 0 N.D. 0 N.D. 01,2,3,4,6,7,8-HpCDF 0.01 N.D. 0 N.D. 0 N.D. 0 1,2,3,4,7,8,9-HpCDF 0.01N.D. 0 N.D. 0 N.D. 0 OCDF 0.0003 N.D. 0 N.D. 0 N.D. 0 total PCDDs +PCDFs 0 0 0 Coplanar Non- 3,3′,4,4′-TeCB (#77) 0.0001  0.82 0.0000820.54 0.000054  0.45 0.000045 PCB ortho 3,4,4′,5-TeCB (#81) 0.0003 N.D. 0N.D. 0 N.D. 0 forms 3,3′,4,4′,5-PeCB (#126) 0.1 N.D. 0 N.D. 0 N.D. 03,3′,4,4′,5,5′-HxCB (#169) 0.03 N.D. 0 N.D. 0 N.D. 0 Mono-2,3,3′,4,4′-PeCB (#105) 0.0003 66   0.0198 19   0.0057 31   0.0093 ortho2,3,4,4′,5-PeCB (#114) 0.0003 N.D. 0 N.D. 0 N.D. 0 forms2,3′,4,4′,5-PeCB (#118) 0.0003 3.1 0.00093 0.45 0.000135 1.2 0.000362′,3,4,4′,5-PeCB (#123) 0.0003 N.D. 0 N.D. 0 N.D. 0 2,3,3′,4,4′,5-HxCB(#156) 0.0003 N.D. 0 N.D. 0 N.D. 0 2,3,3′,4,4′,5-HxCB (#157) 0.0003 N.D.0 N.D. 0 N.D. 0 2,3′,4,4′,5,5′-HxCB (#167) 0.0003 N.D. 0 N.D. 0 N.D. 02,3,3′,4,4,′5,5′-HpCB (#189) 0.0003 N.D. 0 N.D. 0 N.D. 0 total coplanerPCBs 0.020812 0.005889 0.009705 total TEQ(pg-TEQ/g) 0.021 0.006 0.010

EXAMPLE 7

The rectification and column chromatographic processing in the refiningstep after ethyl esterification were verified for possible effects theymight have on the concentration of dioxins. The feedstock oil wassardine oil that was simply diacidified and decolored without subsequentthin-film distillation. This was because use of fats or oils containingmore dioxins would provide greater ease in observing the possibleeffects of the refining process.

The feedstock oil was ethyl esterified using an alkali catalyst. Theresulting ethyl esters were first subjected to a rectification step,thereby collecting fractions containing ethyl esters of C20 fatty acids.Subsequently, a fraction of eicosapentaenoic acid ethyl ester wascollected by ODS column chromatography.

At the respective stages, the contents of dioxins were measured.

Table 9 shows data for the four components that experienced greatervariations (in terms of absolute value and toxicity equivalencyquantity) and the total of dioxins (in toxicity equivalency quantity).The rectification step caused the components #105 and #118 to beconcentrated by far greater degrees than when they were just after theesterification. On the other hand, the content of #77 decreased ratherthan increased. It is assumed that in rectification, ethyl esters ofeicosapentaenoic acid and other fatty acids behaved in a similar way todioxins, so that the dioxins were concentrated as were the ethyl estersof eicosapentaenoic acid and other fatty acids.

Upon column treatment, the content of #77 remained substantially thesame or increased a little whereas the contents of other componentsdropped considerably. It is assumed that in the column treatment, ethylesters and dioxins behaved in a sufficiently different way to enableseparation of the two components.

Therefore, by combining rectification with the column treatment, theconcentrating of the desired ethyl esters and the reduction of dioxinslevels can be achieved simultaneously.

TABLE 9 Non-ortho Non-ortho Mono-ortho Mono-ortho 3,3′4,4′-TeCB3,3′4,4′,5-PeCB 2,3,3′,4,4′-PeCB 2,3′,4,4′,5-PeCB (#77) (#126) (#105)(#118) Total dioxins As esterified 380 pg/g 82 pg/g 4700 pg/g 9700 pg/g(10.62 pg-TEQ/g) (0.038 pg-TEQ/g) (8.2 pg-TEQ/g) (0.47 pg-TEQ/g) (0.97pg-TEQ/g) As rectified 250 170 24000 30000  (23.44) (0.025)  (17) (2.4) (3) As column treated 260    0.69 3700 180 (0.48) (0.026)     (0.069)(0.37)     (0.018)

EXAMPLE 8

Crude sardine oil as feedstock was distilled by SPD as in Example 4 togive an SPD oil, which was ethyl esterified and refined by the sameprocedures as in Example 6 to yield EPA ethyl esters. They were measuredfor the contents of brominated flame retardants. The measurement wascommissioned to the bio-analytical testing company “eurofins”. Theresults are shown in Table 10. The contents of the respective brominatedflame retardants in the feedstock sardine oil had been reduced to lessthan their detection limits. All of BDE-100, BDE-49, BDE-99, BDE-47,BDE-28, BDE-66, and BDE-154 that were contained in the feedstock oil inamounts equal to or greater than their detection limits had been reducedin concentration and, in particular, the concentrations of BDE-100,BDE-49, BDE-99 and BDE-47 were verified to have dropped distinctly.

TABLE 10 Sardine oil SPD oil Ethyl ester CYR11 HBCD(hexabromocyclododecane) Hexabromocyclododecane HBCD (total of a, b, andg forms) <2.0 ng/g <2.0 ng/g <2.0 ng/g CYR16 pbb (polybrominatedbiphenyls) 2,2′,3,3′,4,4′,5,5′,6-nonabromobenzene # BB 206 <0.218 ng/g<0.216 ng/g <0.222 ng/g 2,2′,3,3′,4,4′,5,5′-octabromobenzene # BB 194<0.109 ng/g <0.108 ng/g <0.111 ng/g 2,2′,3,4,4′,5,5′-heptabromobenzene #BB 180 <0.0544 ng/g <0.0541 ng/g <0.0554 ng/g2,2′,4,4′,5,5-hexabromobenzene # 153 <0.0326 ng/g <0.0325 ng/g <0.0333ng/g 2,2′,4,5,5′-pentabromobenzene # 101 <0.0218 ng/g <0.0216 ng/g<0.0222 ng/g 2,2′,5,5′-tetrabromobenzene # 52 <0.0109 ng/g <0.0108 ng/g<0.0111 ng/g decabromobenzene # 209 <0.544 ng/g <0.541 ng/g <0.554 ng/gTotal of bromobenzenes (※) 0.990 ng/g 0.985 ng/g 1.01 ng/g Total ofbromobenzenes (※※) ND ng/g ND ng/g ND ng/g CYR21 FBDE (LR)2,2′,3,3′,4,4,5,5′,6-nonabromodiphenyl ether (BDE-206) <0.218 ng/g<0.216 ng/g <0.222 ng/g 2,2′,3,3′,4,4′,6.6′-octabromodiphenyl ether(BDE-197) <0.109 ng/g <0.108 ng/g <0.111 ng/g2,2′,3,3′4,4′,5,6,6′-nonabromodiphenyl ether (BDE-207) <0.218 ng/g<0.216 ng/g <0.222 ng/g 2,2′,3,4,4′,5,5′,6 -octabromodiphenyl ether(BDE-196) <0.109 ng/g <0.108 ng/g <0.111 ng/g2,2′,3,4,4′,5′-hexabromodiphenyl ether (BDE-138) <0.0326 ng/g <0.0325ng/g <0.0333 ng/g 2,2′,3,4,4′,6,6′-heptabromodiphenyl ether (BDE-184)<0.0544 ng/g <0.0541 ng/g <0.0554 ng/g 2,2′,4,4′,5,6′-hexabromodiphenylether (BDE-154) 0.0384 ng/g <0.0325 ng/g <0.0333 ng/g2,2′,4,4′,6-pentabromodiphenyl ether (BDE-100) 0.0691 ng/g <0.0216 ng/g<0.0222 ng/g 2,2′,4,5′-tetrabromodiphenyl ether (BDE-49) 0.108 ng/g<0.0108 ng/g <0.0111 ng/g 2,2′,4-tribromodiphenyl ether (BDE-17) 0.0118ng/g <0.0108 ng/g <0.0111 ng/g 2,2′,3′,4,4′,5′,6-heptabromodiphenylether (BDE-183) <0.0544 ng/g <0.0541 ng/g <0.0554 ng/g2,2′,3,4,4′-pentabromodiphenyl ether (BDE-85) <0.0218 ng/g <0.0216 ng/g<0.0222 ng/g 2,2′,4,4′,5,5′-hexabromodiphenyl ether (BDE-153) <0 0326ng/g <0.0325 ng/g <0.0333 ng/g 2,2′,4,4′,5-pentabromodiphenyl ether(BDE-99) 0.0994 ng/g <0.0216 ng/g <0.0222 ng/g2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) 0.354 ng/g 0.0131 ng/g<0.0111 ng/g 2,3,3′,4,4′,5′,6-heptabromodiphenyl ether (BDE-191) <0.0544ng/g <0.0541 ng/g <0.0554 ng/g 2,3,3′,4,4′,5-hexabromodiphenyl ether(BDE-156) <0.0326 ng/g <0.0325 ng/g <0.0333 ng/g2,3′,4,4′,6-pentabromodiphenyl ether (BDE-119) <0.0218 ng/g <0.0216 ng/g<0.0222 ng/g 2,3′,4,4′-tetrabromodiphenyl ether (BDE-66) 0.0281 ng/g<0.0108 ng/g <0.0111 ng/g 2,3′,4′,6-tetrabromodiphenyl ether (BDE-71)<0.0109 ng/g <0.0108 ng/g <0.0111 ng/g 2,4,4′-tribromodiphenyl ether(BDE-28) 0.0251 ng/g <0.0108 ng/g <0.0111 ng/g3,3′,4,4′,5-pentabromodiphenyl ether (BDE-126) <0.0218 ng/g <0.0216 ng/g<0.0222 ng/g 3,3′,4,4′-tetrabromodiphenyl ether (BDE-77) <0.0109 ng/g<0.0108 ng/g <0.0111 ng/g Total of octabromodiphenyl ethers (※) 0.218ng/g 0.216 ng/g 0.222 ng/g Total of octabromodiphenyl ethers (※※) NDng/g ND ng/g ND ng/g Decabromodiphenyl ether (BDE-209) <1.09 ng/g <1.08ng/g <1.11 ng/g Total of tetrabromodiphenyl ethers (※) 0.513 ng/g 0.0564ng/g 0.0554 ng/g Total of tetrabromodiphenyl ethers (※※) 0.491 ng/g0.0131 ng/g ND ng/g Total of tribromodiphenyl ethers (※) 0.0369 ng/g0.0216 ng/g 0.0222 ng/g Total of tribromodiphenyl ethers (※※) 0.0369ng/g ND ng/g ND ng/g Total of nonabromodiphenyl ethers (※) 0.435 ng/g0.433 ng/g 0.443 ng/g Total of nonabromodiphenyl ethers (※※) ND ng/g NDng/g ND ng/g Total of bromodiphenyl ethers (※) 2.82 ng/g 2.21 ng/g 2.26ng/g Total of bromodiphenyl ethers (※※) 0.735 ng/g 0.0131 ng/g ND ng/gTotal of hexabromodiphenyl ethers (※) 0.136 ng/g 0.130 ng/g 0.133 ng/gTotal of hexabromodiphenyl ethers (※※) 0.0384 ng/g ND ng/g ND ng/g Totalof heptabromodiphenyl ethers (※) 0.163 ng/g 0.162 ng/g 0.166 ng/g Totalof heptabromodiphenyl ethers (※※) ND ng/g ND ng/g ND ng/g Total ofpentabromodiphenyl ethers (※) 0.234 ng/g 0.108 ng/g 0.111 ng/g Total ofpentabromodiphenyl ethers (※※) 0.169 ng/g ND ng/g ND ng/g CVR26 TBBPA(tetrabromobisphenol A) tetrabromobisphenol A (※※) <0.538 ng/g <0.544ng/g <0.544 ng/g (※) = (calculated in terms of quantification limit)(※※) = (values less than the quantification limit are deleted)

INDUSTRIAL APPLICABILITY

According to the present invention, the amounts of environmentalcontaminants, especially dioxins and brominated flame retardants, thatare contained in fats or oils that comprise highly unsaturated fattyacids as constituent fatty acid, as exemplified by fish oils containingEPA and DHA can be markedly reduced, making it possible to provide fatsor oils having this feature. The products thus obtained are suitable foruse in feeds, foods, supplements, medicines, and the like.

1. A method of producing a highly unsaturated fatty acid or a highlyunsaturated fatty acid ethyl ester with reduced contents ofpolychlorinated dibenzoparadioxins (PCDDs), polychlorinateddibenzofurans (PCDFs) and coplanar PCB (Co-PCBs), the method comprisingthe steps of: a) removing free fatty acids and environmental pollutantsby thin-film distillation from a feedstock oil containing highlyunsaturated fatty acids as constituent fatty acids; b) ethyl esterifyingthe highly unsaturated fatty acids present as constituent of fat or oilcontained in the product of step (a); c) refining the product of step(b) by rectification and column chromatography, wherein a concentrationof the highly unsaturated fatty acid ethyl ester in the sum of fattyacid ethyl esters is at least 80 area %.
 2. The method according toclaim 1, wherein amount of a brominated flame retardant in the highlyunsaturated fatty acid or the highly unsaturated fatty acid ethyl esteris also reduced.
 3. The method according to claim 1, wherein theconcentration of the highly unsaturated fatty acid ethyl ester in thesum of fatty acid ethyl esters is at least 85 area %.
 4. The methodaccording to claim 1, wherein the feedstock oil containing highlyunsaturated fatty acids as constituent fatty acids is fish oil, krilloil, marine mammal oil or microorganism oil.
 5. The method according toclaim 1, wherein the thin-film distillation is carried out at atemperature of 200-270° C.
 6. The method according to claim 1, whereinthe thin-film distillation is carried out at a pressure of 5 Pa orlower.
 7. The method according to claim 1, wherein the thin-filmdistillation is carried out at a flow rate of 20-200 (kg/h)/m².
 8. Themethod according to claim 1, wherein the thin-film distillation ismolecular distillation or short path distillation.
 9. The methodaccording to claim 1, wherein rectification is performed in three ormore distillation columns.
 10. The method according to claim 1, whereinrefining by column chromatography uses column chromatography ofreverse-phase distribution type.
 11. The method according to claim 1,wherein in step (c) the concentration of the highly unsaturated fattyacid ethyl ester in the sum of fatty acid ethyl esters is at least 90area %.
 12. The method according to claim 1, wherein in step (c) theconcentration of the highly unsaturated fatty acid ethyl ester in thesum of fatty acid ethyl esters is at least 95 area %.
 13. The methodaccording to claim 1, wherein in step (c) the concentration of thehighly unsaturated fatty acid ethyl ester in the sum of fatty acid ethylesters is at least 80 area % and at most 97 area %.
 14. The methodaccording to claim 1, wherein the method further comprises the step of:d) producing the highly unsaturated fatty acid by hydrolyzing the highlyunsaturated fatty acid ethyl ester of step (c).